Charif et al. Molecular Neurodegeneration (2021) 16:12 https://doi.org/10.1186/s13024-021-00431-w

SHORT REPORT Open Access Dominant mutations in MIEF1 affect mitochondrial dynamics and cause a singular late onset optic neuropathy Majida Charif1,2†, Yvette C. Wong3†, Soojin Kim3, Agnès Guichet4, Catherine Vignal5, Xavier Zanlonghi6, Philippe Bensaid7, Vincent Procaccio1,4, Dominique Bonneau1,4, Patrizia Amati-Bonneau1,4, Pascal Reynier1,4, Dimitri Krainc3 and Guy Lenaers1*

Abstract Inherited optic neuropathies are the most common mitochondrial diseases, leading to neurodegeneration involving the irreversible loss of retinal ganglion cells, optic nerve degeneration and central visual loss. Importantly, properly regulated mitochondrial dynamics are critical for maintaining cellular homeostasis, and are further regulated by MIEF1 (mitochondrial elongation factor 1) which encodes for MID51 (mitochondrial dynamics protein 51), an outer mitochondrial membrane protein that acts as an adaptor protein to regulate mitochondrial fission. However, dominant mutations in MIEF1 have not been previously linked to any human disease. Using targeted sequencing of genes involved in mitochondrial dynamics, we report the first heterozygous variants in MIEF1 linked to disease, which cause an unusual form of late-onset progressive optic neuropathy characterized by the initial loss of peripheral visual fields. Pathogenic MIEF1 variants linked to optic neuropathy do not disrupt MID51’s localization to the outer mitochondrial membrane or its oligomerization, but rather, significantly disrupt mitochondrial network dynamics compared to wild-type MID51 in high spatial and temporal resolution confocal microscopy live imaging studies. Together, our study identifies dominant MIEF1 mutations as a cause for optic neuropathy and further highlights the important role of properly regulated mitochondrial dynamics in neurodegeneration. Keywords: Mitochondria dynamics, Mitochondrial disease, MIEF1, Mid51, Dominant optic atrophy (DOA), Inherited optic neuropathy (ION), Peripheral visual field, Neurodegeneration

Main text mutations. In dominant optic atrophy (DOA, Inherited Optic Neuropathies (ION) are the most com- MIM165500), it is now well established that mutations mon mitochondrial diseases, leading to the irreversible in genes involved in mitochondrial dynamics such as loss of retinal ganglion cells, optic nerve degeneration OPA1 (MIM#605290) are the main cause of the disease. and central visual loss [1, 2]. They can be maternally More than 400 distinct pathogenic variants have been transmitted by the mitochondrial genome in the case of described in OPA1 [3–6], affecting the pro-fusion activ- Leber hereditary optic neuropathy (LHON, ity of this large dynamin-related GTPase resulting in de- MIM#535000), or by mono or bi-allelic autosomal fective mitochondrial fusion [7, 8]. The implication of mitochondrial dynamics in DOA was further supported MFN2 * Correspondence: [email protected] by the identification of dominant mutations in †Majida Charif and Yvette C. Wong contributed equally to this work. (MIM#608507) [9] and OPA3 (MIM#606580) [10], two 1 Université d’Angers, MitoLab team, UMR CNRS 6015 - INSERM U1083, Unité additional genes acting on mitochondrial dynamics. MitoVasc, Angers, France Full list of author information is available at the end of the article

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More recently, we and others described cohorts of in- encephalopathy associated with optic atrophy and per- dividuals affected by DOA caused either by mutations in ipheral neuropathy [21], while no mutation has been re- SPG7 (MIM#602783) or in AFG3L2 (MIM#604581) ported for optic atrophy in MIEF1 or MIEF2, which [11–13], two genes encoding mAAA-proteases acting on encode for MID51 and MID49, respectively. mitochondrial fusion through the indirect control of Together, these observations prompted us to screen all OPA1 processing, and also involved in recessive heredi- known genes involved in mitochondrial dynamics in mo- tary spastic paraplegia type 7 [14] and dominant spino- lecularly undiagnosed ION cases. Here, we identify the cerebellar ataxia 28 [15], respectively. In addition, homo- first heterozygous pathogenic disease-causing mutations zygous mutations in YME1L1 (MIM#617302), which also in MIEF1, found in individuals with non-syndromic late- disrupt OPA1 processing have been identified in individ- onset ION characterized by initial loss of peripheral vis- uals with optic atrophy and mitochondrial disorders ual fields. Importantly, ION disease-linked MIEF1 vari- [16]. Interestingly, DNM1L mutations resulting in exces- ants affect mitochondrial network dynamics, and sive mitochondrial fusion have also been identified in ultimately highlight the crucial role of properly regulat- patients with a similar ophthalmological presentation ing this pathway in optic nerve physiology. [17] (MIM603850), thus providing evidence that alter- Two hundred individuals in France affected by an ION ations of both fusion and fission compromise retinal were included in this study. None of the individuals in- ganglion cell survival. DNM1L encodes DRP1, another cluded had a molecular diagnosis after screening for dynamin-related GTPase which requires a set of adaptor pathogenic variants in OPA1, OPA3 and WFS1 exonic proteins including MID51, MID49, and MFF to be re- sequences and LHON-associated mtDNA mutations. In- cruited on the mitochondrial outer membrane to exert dividuals were analyzed using a targeted resequencing and regulate its pro-fission activity [18–20]. So far, only panel of 22 genes, including those already published as biallelic nonsense MFF mutations (MIM#614785) have causing ION, and genes involved in mitochondrial dy- been identified in individuals with a severe namics (Supplementary Table 1). After eliminating

Fig. 1 Characterization of inherited optic neuropathy patients with dominant MIEF1 mutations. a Eye fundus of the right (RE) and left (LE) eye exhibiting severe optic disk pallor in MIEF1 individuals with inherited optic neuropathy (Patient 1 (left 2 panels); Patient 2 (right 2 panels)). b Evaluation of the visual fields revealed peripheral visual field defects in MIEF1 individuals with inherited optic neuropathy (Patient 1 (left 2 panels); Patient 2 (right 2 panels)). c Optical coherence tomography (OCT) recordings of the retinal nerve fiber layer (RNFL) at the papilla of the right (RE, left panel) and left (LE, right panel) eye from Patient 2 with inherited optic neuropathy Charif et al. Molecular Neurodegeneration (2021) 16:12 Page 3 of 9

frequent (allele frequency > 1/10.000) and non- defect in the second individual, together with variable pathogenic variants, according to prediction tools (Sift, central visual alterations (Fig. 1b). In both individuals, Polyphen, and Mutation-Taster), we identified two indi- the disease progressed to poor vision (Patient 1: RE: 1/ viduals (Fig. 1) harboring a MIEF1 heterozygous variant, 20, LE: 18/20; Patient 2: RE: hand moving, LE: 4/20), which were confirmed by Sanger sequencing. The first asymmetrically in the first one and symmetrically in the individual harbored a c.718 T > A variant, not referenced second one. Accordingly, optical coherence tomography in any database, and the second individual harbored a (OCT) scanning disclosed severe retinal nerve fiber layer c.436C > T variant, referred to as rs778124994, with a loss in all quadrants in the second individual (Fig. 1c). frequency of 1.99e-5 in GnomAD database. Both vari- No additional neurological or systemic symptoms were ants were predicted to be damaging by the Sift and Poly- observed in either patient. phen programs and disease causing by Mutation Taster Thus, the optic atrophy that we report here is an ori- (Fig. 2a). These variants lead to the p.Y240N and the ginal ophthalmological presentation based on three dis- p.R146W amino-acid changes in MID51, respectively, criminating criteria. First, the visual loss was noticed with p.Y240N located in a DRP1 binding domain, and during adulthood, rather than during the first two de- p.R146W located in another domain conserved within cades, as commonly observed in other DOA, although a MID49, but without known function (Fig. 2b, c). latent alteration of the peripheral visual fields might The two middle-aged women, 55 and 47 years old, car- have preceded the loss of visual acuity by several years, rying MIEF1 mutations were initially referred to oph- as observed in one individual. Secondly, both patients thalmology departments for a sudden painless visual complained of a rather sudden painless loss of visual acuity impairment (see Methods for detailed patient de- acuity, similar to those observed in maternally inherited scriptions). At initial assessments, neither of the two in- LHON, although without optic disk elevation or edema, dividuals had symptoms or immunological profiles which is distinct from what is generally observed for compatible with Glaucoma, Neuro Myelitis Optica or other autosomal optic neuropathies, except for a recent myelin oligodendrocyte glycoprotein optic neuritis. At report presenting a LHON-like patient with bi-allelic fundus examination, both individuals had normal retina, NDUFS2 mutations [22]. Thirdly, both cases examined but presented pale and moderately excavated optic disks here showed that the disease progressed from the per- (Fig. 1a). Importantly, both had an alteration of periph- ipheral to the central visual field, still preserving some eral visual fields, with a clear-cut bilateral superior field visual acuity in the left eye of the first patient. This is in

Fig. 2 Genotype of dominant MIEF1 variants in individuals with inherited optic neuropathy. a Genetic analysis identified dominant MIEF1 mutations: heterozygous mutation c.718 T > A in exon 6 of MIEF1 localized at position 240 of the MID51 protein, leading to an amino acid exchange of tyrosine to asparagine (p.Y240N; Patient 1); and heterozygous mutation c.436C > T in exon 5 of MIEF1 localized at position 416 of the MID51 protein, leading to an amino acid exchange of arginine to tryptophan (p.R146W; Patient 2). Additional variant characterization according to the Sift, PolyPhen-2 and Mutation Taster scores are shown. b Alignment of MID51 (NM_019008.5) and MID49 (NM_139162.4) protein sequences around the mutated amino acids (p.R146W (green) and p.Y240N (red)) showing conservation of R146 in MID51. c Localization of the amino acid changes (p.R146W (green) and p.Y240N (red)) on the MID51 protein. MID51 contains a transmembrane domain (TM) mediating its insertion into the outer mitochondrial membrane, and a DRP1 binding region in which Y240 is located Charif et al. Molecular Neurodegeneration (2021) 16:12 Page 4 of 9

contrast with all the reports of ION cases, in which the p.R146W mutants resulted in significantly decreased alteration of the visual field occurs and evolves from the mitochondrial fusion events and disrupted mitochon- center to the periphery. drial network dynamics, as compared to wild-type To assess the pathogenicity of the MIEF1 variants, we MID51 (Fig. 3j-m). Together, these results show that expressed wild-type and both mutants (p.Y240N and MIEF1 mutations linked to optic neuropathy preferen- p.R146 W) of MID51 in HeLa cells. MID51 is embedded tially disrupt the ability of MID51 to regulate mitochon- in the outer mitochondrial membrane, where it homo- drial fission/fusion dynamics. oligomerizes to regulate mitochondrial fission [18, 19]. In summary, combining our clinical, genetic and Thus, we first examined whether p.R146W and p.Y240N pathophysiological data, we identify the first known mutants displayed a mitochondrial localization, using dominant MIEF1 mutations linked to a human disease, high spatial and temporal confocal microscopy in live resulting in a specific ophthalmological neurodegenera- cells. Expression of wild-type MID51 robustly localized tive disease. MID51 is a key regulator of mitochondrial to mitochondria (mEmerald-Mito) (Fig. 3a, b), consistent dynamics [18, 19, 23–27]. Our findings that Mid51 optic with its reported localization within the outer mitochon- neuropathy-linked variants disrupt mitochondrial fis- drial membrane [18, 19]. This was also true for mutant sion/fusion dynamics but not its localization or MID51 p.Y240N and p.R146W proteins, which also lo- oligomerization are consistent with the fact that p.Y240 calized to the mitochondrial network in live cells (Fig. is a residue located in the loop region (residues 238– 3a, b), demonstrating that these MIEF1 missense vari- 242) critical for DRP1 binding [28, 29] which regulates ants linked to optic neuropathy do not alter MID51’s mitochondrial fission [30]. In contrast, neither disease- mitochondrial localization. linked mutations (p.Y240N or p.R146W) are located in We next investigated whether MIEF1 variants dis- MID51’s transmembrane domain which mediates its rupted the ability of MID51 to self-oligomerize. To test outer mitochondrial membrane localization (residues 1 this, we immunoprecipitated myc-tagged wild-type and to 48) [19], or in previously characterized residues medi- mutant MID51 proteins, and analyzed their ating Mid51’s oligomerization [29]. Together, our work oligomerization patterns. Wild-type MID51 formed should prompt the molecular screening of MIEF1 in monomers as well as oligomers, which consisted of di- ION individuals with severe alterations of the peripheral mers, tetramers, and very few high molecular weight visual field, and further emphasizes the crucial role for (HMW) species (Fig. 3c). Similarly, we found that both properly regulated mitochondrial dynamics in neurode- mutant proteins MID51 p.Y240N (Fig. 3d-f) and MID51 generative diseases. p.R146W (Fig. 3g-i) also showed similar oligomerization patterns to wild-type MID51 (Fig. 3c), with similar ratios Methods of each oligomeric species to monomer levels. Thus, Population screening these MIEF1 missense variants linked to optic neur- Two hundred individuals in France (112 males and 88 opathy do not significantly disrupt the oligomerization females) with an inherited optic neuropathy were in- of MID51. cluded in this study. Their DNA samples were collected Finally, as MID51 is a known regulator of mitochon- at the Department of Biochemistry and Genetics from drial fission dynamics [18, 19], we studied whether mito- the Angers University Hospital (France), which is a na- chondrial fission/fusion dynamics were perturbed by the tional centre for the molecular diagnosis of ION. All pa- MIEF1 variants. Expression of wild-type MID51 has pre- tients presented with an isolated dominant or recessive viously been shown to inhibit mitochondrial fission, optic atrophy, and were diagnosed by an ophthalmolo- resulting in increased mitochondrial fusion events [19]. gist from the French National Reference Centres for We analyzed the rate of mitochondrial fusion events in Rare Blinding Diseases. live cells, using a photo-activatable mitochondrial matrix probe (mito-PAGFP) in high spatial and temporal reso- Subjects lution confocal microscopy live imaging studies. A sub- The first affected individual aged 55 is from Maghreb or- population of mitochondria was selectively igins (Patient 1). In 2002, she noticed a faint visual prob- photoactivated using a 405 nm laser, and the rate of fu- lem on the right eye, with a reported best visual acuity sion events with the surrounding mitochondria was ana- (bva) of 10/20 at first examination. In 2015, she com- lyzed by measuring the increase in fluorescence intensity plained of an acute painless visual loss, affecting mainly over time at a distal region of 10 μm from the site of the peripheral visual field of both eyes, and also the cen- photoactivation (Fig. 3j). Consistent with previous re- tral visual field of the right eye (bva:1/20), but not that ports, we found that wild-type MID51 expression in- of the left eye (bva: 18/20) (Fig. 1b, left). Visual evoked creased the rate of fusion events (Fig. 3j-m) compared to potentials were strongly affected for both eyes, while control cells. In contrast, both MID51 p.Y240N and scotopic and photopic electroretinograms (ERGs) were Charif et al. Molecular Neurodegeneration (2021) 16:12 Page 5 of 9

Fig. 3 Inherited optic neuropathy MIEF1 mutations do not disrupt MID51 localization or oligomerization, but preferentially affect its ability to regulate mitochondrial network dynamics. a and b Representative live-cell confocal images and quantification of mCherry-tagged MID51 (WT; p.Y240N; p.R146W) showing MID51 localization to mitochondria (mEmerald-Mito). (n = 100 cells from 3 experiments/ per condition). Scale bar, 10 μm (inset, 1 μm). c-i Representative immunoblot (α-myc) and quantification of MID51 oligomerization into dimer, tetramer, or high molecular weight (HMW) species from IP (myc) of myc-tagged MID51 (WT; p.Y240N; p.R146W) and control (−-). Mutants MID51 p.Y240N (d-f) and MID51 p.R146W (g-i) show similar oligomerization compared to wild-type MID51 (WT). (n = 3 experiments/per condition). j-m Representative confocal live-cell images and traces (j and k) and analysis (l and m) of mito-PAGFP fluorescence intensity in distal region (10 μm from the site of mitochondrial photoactivation) in high spatial and temporal resolution confocal microscopy live imaging studies, showing increased mitochondrial fusion (white arrows) in wild-type MID51 (WT) condition which is not observed in mutant MID51 p.Y240N and p.R146W conditions. (n = 8 cells (control); n = 9 cells (WT); n = 16 cells (p.Y240N); n = 30 cells (p.R146W), from 3 experiments/per condition). Scale bar, 5 μm. Data are means ± s.e.m. (N.S. = not significant; **P < 0.01; ***P < 0.001; unpaired two-tailed t test (d-i); ANOVA with Tukey’s post-hoc test (b, l, m)) Charif et al. Molecular Neurodegeneration (2021) 16:12 Page 6 of 9

normal. Eye fundus examination revealed a normal ret- WFS1 exonic sequences and the three primary LHON ina, while the optic disks appeared pale and moderately mtDNA mutations. Negative cases were analyzed by tar- excavated (Fig. 1a, left). Blood analyses, and in particular geted resequencing panel of 22 genes (Supplementary vitamin B9 and B12 concentrations and Lyme and Table 1) including published ION genes and candidate Treponema serology, were normal, but she had higher genes involved in mitochondrial dynamics (FIS1, MFF, sedimentation rate (44 mm, with normal Protein C Re- MFN1, MIEF1, MIEF2, OMA1 and YME1L1), but not active concentration). She also suffered from sero- known to be involved in ION. negative rheumatoid polyarthritis treated by ARAVA The amplicon library targeting the exons from the 22 (20 mg/day), while no peripheral neuropathy was genes (Supplementary Table 1) was designed with the noticed. Ion AmpliSeq Designer (http://ampliseq.com). The The second individual aged 47 was born in Egypt (Pa- Qubit dsDNA High Sensitivity Assay Kit (Thermo Fisher tient 2). She did not complain of any visual defect during Scientific) was used to quantify DNA for NGS library her youth. In 2002, 1 month after an uneventful cesarean construction. Library preparation for each sample was delivery, she complained of a painless visual loss first on performed using Ion AmpliSeq technologies, sequencing the right eye (bva: 3/20), then on the left eye (bva: 10/ was undertaken using 540 ChIPs on Ion S5™ Sequencer 20), while the visual field examination disclosed severely using barcoded samples, and sequencing data was proc- narrowed isopters, in particular on the superior quad- essed using our own dedicated bioinformatics pipeline as rant, reflecting a bilateral superior altitudinal defect (Fig. described elsewhere [31, 32]. Within the design regions, 1b, right). She presented with pale and excavated optic 95% of bases had coverage over 100X, indicating that disks (Fig. 1a, right) with normal intra-ocular pressure sufficient coverage was achieved to enable high variant (IOP: RE: 9 and LE: 10 mmHg). Her bilateral optic neur- detection sensitivity. Sanger sequencing was performed opathy progressed to 2/20 and 8/20 in 2012, to moving in parallel for regions that were not or poorly covered by hand and 4/20 in 2015, respectively for the right and left ChIP sequencing. eye. VEP (visual evoked potential) were strongly affected, For each patient, we screened for causative variants while the retina was normal, except for the presence of using the following prioritization strategy. Firstly, we macular microcysts. OCT (optical coherence tomog- checked reported pathogenic variants in ION published raphy) examination revealed collapsed RNFL (retinal genes, then we looked for novel loss-of-function (LOF) nerve fiber layer) in all quadrants in both eyes (Fig. 1c). variants (stop-gain, frameshift and splicing) or novel Anti-NMO and anti-MOG serology were negative. Brain missense variants and finally, we selected pathogenic MRI and ENT examinations were normal. She suffers variants in candidate unpublished genes. All dominant from hypothyroidism, hypercholesterolemia and iron de- variants were either absent or had a minor allele fre- ficiency, but has no diabetes, epilepsy or neuro-muscular quency (MAF) threshold of < 0.0001 in public databases disorder. Biotinidase activity was normal. (GnomAD), while recessive variants were considered In both individuals, Sanger sequencing did not reveal with a MAF threshold < 0.005. For familial cases, we mutations in OPA1, OPA3, WFS1, or mtDNA LHON specifically selected the variants that matched the inher- mutations. In addition, SNP array analyses revealed no itance pattern predicted from the pedigrees [32]. The chromosomal abnormality, no copy number variation two candidate pathogenic variants in MIEF1 were vali- and no large region of homozygosity that might have in- dated by Sanger sequencing. dicated a consanguinity.

Standard protocol approvals, registrations, and patient Reagents consents The following plasmids were obtained from Addgene: Written informed consent to perform genetic analyses mEmerald-Mito-7 was a gift from Michael Davidson was obtained from each subject involved in this study, (Addgene plasmid # 54160; http://n2t.net/addgene:5416 according to protocols approved by the Ethical Commit- 0; RID:Addgene_54,160) [33], mito-PAGFP was a gift tees of the different Institutes involved in this study, and from Richard Youle (Addgene plasmid # 23348; http:// in agreement with the Declaration of Helsinki (Institu- n2t.net/addgene:23348; RRID:Addgene_23,348) [34], tional Review Board Committee of the University Hos- mApple-TOMM20-N-10 was a gift from Michael David- pital of Angers, Authorization number: AC-2012-1507). son (Addgene plasmid # 54955; http://n2t.net/ addgene:54955; RRID:Addgene_54,955). C-terminal Genetic analysis mCherry-tagged MID51 (wildtype (WT); p.Y240N; Genomic DNA was extracted from peripheral blood cells p.R146W) and C-terminal myc-tagged MID51 (wildtype from a cohort of individuals in France with ION and (WT); p.Y240N; p.R146W) were generated by Vector- screened for pathogenic variants in OPA1, OPA3 and Builder. The following antibodies were also used: rabbit Charif et al. Molecular Neurodegeneration (2021) 16:12 Page 7 of 9

Myc-tag (Cell Signaling, 2272S) and mouse Myc-Tag Mitochondrial fusion dynamics were analyzed as the (9B11) (Cell Signaling, 2276). mito-PAGFP fluorescence intensity at a distal region (10 μm from the site of photoactivation) at 1 min and 5 Cell culture and transfection min post-photoactivation, and subsequently normalized HeLa cells (ATCC) were cultured in DMEM (Gibco; to the intensity at t = 0 min. Example traces of mito- 11995–065) supplemented with 10% (vol/vol) FBS, 100 PAGFP max intensity (Fig. 3k) are the average of 3 inde- units per ml penicillin, and 100 μg/ml streptomycin. All pendent examples (n = 3 cells from 3 experiments/ per cells were maintained at 37 °C in a 5% CO2 incubator condition). The mito-PAGFP fluorescence intensity used and verified by cytochrome c oxidase subunit I (COI) was the maximum intensity in a 4 μm×4μm area in the and short tandem repeat (STR) testing, and were nega- distal region. Immunoblots were quantified using ImageJ tive for mycoplasma contamination. Cells were trans- (NIH). fected using Lipofectamine 2000 (Invitrogen). For live imaging, cells were grown on glass-bottom culture Statistical analysis, graphing and figure assembly dishes (MatTek; P35G-1.5-14-C). Data were analyzed using unpaired two-tailed t test (for two datasets) and one-way ANOVA with Tukey’s post Confocal microscopy hoc test (for multiple datasets), and statistics are shown Confocal images were acquired on a Nikon A1R con- comparing MID51 (WT) to MID51 mutants. Data pre- focal microscope with GaAsp detectors using a Plan sented are means ± s.e.m. with n ≥ 3 independent experi- Apo λ 100 × 1.45 NA oil immersion objective (Nikon) ments (biological replicates) per condition. Statistics and using NIS-Elements (Nikon). Live cells were imaged in a graphing were performed using Prism 7 (GraphPad) temperature-controlled chamber (37 °C) at 5% CO2 at 1 software. All videos and images were assembled using frame every 2–3 s. For mito-PAGFP experiments, the ImageJ 1.51j8 (NIH) and Illustrator CC (Adobe). matrix of a subpopulation of mitochondria were select- ively labeled by localized photoactivation, using a 405 Supplementary Information nm laser (100% for 4 s) in cells transfected with photoac- The online version contains supplementary material available at https://doi. tivatable mitochondrial matrix marker mito-PAGFP and org/10.1186/s13024-021-00431-w. either control (mApple-Tom20) or mCherry MID51 Additional file 1: Supplementary Table 1. Targeted sequencing panel (WT, Y240N or R146W). Fluorescence intensity at a dis- of ION genes and genes involved in mitochondrial dynamics. tal region of 10 μm from the site of photoactivation was analyzed. Abbreviations AFG3L2: AFG3 like matrix AAA peptidase subunit 2; Anti-MOG: Anti-myelin Immunoprecipitation oligodendrocyte glycoprotein; Anti-NMO: Anti-neuromyelitis optica; To examine MID51 oligomerization, HeLa cells were DNA: Deoxyribonucleic acid; DNM1L/DRP1: Dynamin-related protein 1; DOA: Dominant optic atrophy; ENT: Ear, nose and throat; Fis1: Mitochondrial transfected for 24 h with myc-tagged MID51 (WT, fission 1 protein; ION: Inherited Optic Neuropathies; IOP: Intra-ocular Y240N or R146W) or not transfected (control). Cells pressure; LHON: Leber hereditary optic neuropathy; MFF: Mitochondrial were lysed in EBC buffer (Boston Bioproducts; C14–10) fission factor; MFN2: Mitofusin 2; MID49: Mitochondrial dynamics protein 49; MID51: Mitochondrial dynamics protein 51; MIEF1: Mitochondrial elongation with cOmplete™ Protease Inhibitor Cocktail (Roche; 11, factor 1; MIEF2: Mitochondrial elongation factor 2; mito-PAGFP: Mitochondrial 873,580,001) and sonicated. Lysates were immunopreci- photoactivatable green fluorescent protein; MRI: Magnetic resonance pitated using Protein G-coupled Dynabeads (Invitrogen; imaging; NDUFS2: NADH:Ubiquinone Oxidoreductase Core Subunit S2; OCT: Optical coherence tomography; OPA1: Optic Atrophy Protein 1; 100003D) incubated in anti-myc antibody (ms), and OPA3: Optic Atrophy Protein 3; RNFL: Retinal nerve fiber layer; SPG7: Spastic washed in EBC buffer. Equal total protein levels of im- paraplegia 7; WFS1: Wolframin; WT: Wild type; YME1L1: YME1 Like 1 ATPase munoprecipitate were analyzed by SDS-PAGE and Acknowledgements Western blot according to standard protocols using anti- Fluorescence imaging work was performed at the Northwestern University myc antibody (Rb). Measured band intensities of immu- Center for Advanced Microscopy, generously supported by a NCI CCSG P30 noprecipitated MID51 for oligomeric species (dimer, CA060553 grant awarded to the Robert H. Lurie Comprehensive Cancer Center. tetramer or high molecular weight (HMW)) were nor- malized to monomeric MID51, and further normalized Authors’ contributions to MID51 (WT), and expressed as the ratio of MID51 YCW and GL: Conception and design of the study. CV, XZ, PB and DB: Clinical investigation and phenotyping. MC, YCW, SK, AG, VP, PA, PR and DK: oligomer/monomer for each condition. Acquisition and analysis of data. MC, YCW and GL: Drafting and editing figures and manuscript. All authors read and approved the final manuscript. Image analysis MID51 localization to mitochondria was quantified as Funding This work was supported by the Université d’Angers, CHU d’Angers, the the percentage of cells that had MID51 (mCherry- Région Pays de la Loire, Angers Loire Métropole, the Fondation Maladies tagged) colocalized with mitochondria (mEmerald-mito). Rares, the Fondation VISIO, Kjer-France, Ouvrir Les Yeux, Retina France, Charif et al. Molecular Neurodegeneration (2021) 16:12 Page 8 of 9

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